The innate immune systems of humans and animals comprise a number of different mechanisms by which cells in these organisms recognize and respond to a variety of different pathogen-associated molecular patterns (PAMPS) on foreign substances or damage-associated (or danger-associated) molecular patterns due to components released due to damaged cells or stress signals from damaged cells (DAMPS). Pattern recognition receptor (PRR) proteins, including membrane-bound toll-like receptors (TLRs) and cytoplasmic NOD-like receptors (NLRs), recognize a variety of different ligands as foreign, damaged, or non-self (e.g., pathogen-associated or damage-associated) and activate one or more innate immune response pathways that function to defend the organism. In some cases, the innate immune response pathway may result in damage or death of the cell after binding of a ligand on a foreign substance to the PRR of the cell. For example, a type I interferon (IFN) response is induced upon binding of double-stranded RNA (dsRNA) to toll-like receptor 3 (TLR3) or binding of a Gram-negative lipopolysaccharide (LPS) to TLR4, and these IFN responses can inhibit protein translation in the cell and induce many other innate immune response pathways that result in damage to the cell, or, if sustained over time (e.g., by repeated exposure to the foreign substance comprising dsRNA or LPS over multiple days), result in death of the cell.
The Interferon family of cytokines is one key component of the innate immune response to both bacterial and viral infection. Interferons were discovered more than 50 years ago as biological agents that inhibited the replication of influenza virus (Isaacs and Lindenmann, 1957). Interferons are designated type I-III based on the receptor complex they signal through. Type I IFNs, which comprise 13 IFNα subtypes, IFNβ, IFNκ, IFNε, IFNo, IFNτ and IFNδ, engage the ubiquitously expressed IFNAR (IFNα receptor) complex that is composed of the IFNAR1 and IFNAR2 subunits. The functions of Type I IFNs are well characterized and known to be essential for mounting a robust anti-viral response (Muller et al., 1994). Type II IFNs consist of the single IFNγ protein that binds the IFNγ receptor (IFNGR) complex. IFNγ secretion functions primarily to inhibit pathogens other than viruses. Type III IFNs consist of 3 IFNλs and signal through IFNLR1 and IL-10R2. At present, not much is known regarding type III IFNs other than that they are known to regulate an antiviral response and may be the ancestral type I IFNs (Levraud et al., 2007).
Elevated type I IFN levels have been shown to play major roles in the disease states in autoimmune disorders such as psoriasis and systemic lupus erythematosus (SLE) (Hua et al., 2006; Kirou et al., 2005; Nestle et al., 2005). Neutralization of type I IFNs or type I IFN receptors with anti-interferon pathway-specific antibodies have been shown to reduce psoriasis and SLE disease progression (Nestle et al., 2005; Yao et al., 2009).
Viral infections initiate an innate immune response in infected cells resulting in a cascade of intracellular events, ultimately resulting in the secretion of interferons. Triggering the innate immune response can result in apoptosis of the cell or inhibition or repression of protein synthesis. Immunorecognition of viruses is dependent on detection of viral nucleic acids by PPRs, including TLRs. TLR3 activates an innate immune response by recognizing and binding to virally-derived dsRNA (Alexopoulou et al., 2001; Wang et al., 2004). TLR9 is activated by DNA containing unmethylated CpG motifs, found in viral and bacterial DNA (Hemmi et al., 2000). Single-stranded RNAs (ssRNA) and small interfering RNA (siRNAs) are recognized by TLR7 and TLR8 (Diebold et al., 2004; Heil et al., 2004; Hemmi et al., 2002; Judge and MacLachlan, 2008). TLR4 activates an innate immune response by recognizing and binding to LPS of Gram-negative bacteria. Innate immune responses induced by different foreign substances activating different TLRs can be mediated, at least in part, through common signaling pathways. For example, activation of both TLR3 and TLR4 trigger signaling pathways that result in production of type I interferons (IFNs).
Vaccinia virus (VV), a cytoplasmic DNA virus in the poxvirus family, encodes a set of intracellular proteins or soluble cytokine binding proteins that enhance virus virulence. VV intracellular E3L protein, an inhibitor of interferon induction, binds to dsRNA and prevents the activation of the IFN-induced protein kinase PKR (Chang et al., 1992). VV intracellular K3L protein binds competitively to PKR and blocks the phosphorylation and inactivation of host eIF-2α (Beattie et al., 1991). VV also encodes a secreted IFNα/β receptor that is encoded by the B18R gene (Colamonici et al., 1995; Symons et al., 1995). This B18R gene encodes a secreted glycoprotein that binds to and inhibits the function of type I interferons (IFNα/β), while not binding nor inhibiting type II interferons (IFNγ) (Symons et al., 1995). Vaccinia strains lacking functional B18R show much lower levels of viral virulence demonstrating the importance of inhibiting type I interferons during viral infection (Colamonici et al., 1995; Symons et al., 1995).
In vitro-transcribed mRNA made from SP6, T7 or T3 RNA polymerases have been shown to function in countless studies when used for direct injection into Xenopus laevis (frog) or Danio rerio (zebrafish) ooyctes as well as for transfection into mammalian cells in culture. It is well established that in vitro transcription using T7 RNA polymerase can result in the generation of some dsRNA in addition to the desired ssRNA (Cazenave and Uhlenbeck, 1994; Triana-Alonso et al., 1995). Introduction of viral dsRNA or the synthetic dsRNA cohomopolymer polyinosinic-polycytidylic acid (polyI:C) results in the activation of a TLR3-mediated innate immune response (Alexopoulou et al., 2001; Schulz et al., 2005). Similarly, introduction of in vitro-transcribed mRNA or dsRNA into mammalian cells results in the activation of TLR3-mediated innate immune response, signified by the production of type I interferons (Kariko et al., 2004). Addition of recombinant B18R protein to the media of cells transfected with in vitro-transcribed mRNAs reduces mRNA-induced toxicity, presumably through the inhibition of interferon activity (Angel and Yanik, 2010; Warren et al., 2010); and U.S. Patent Application No. 20100273220)